Probing The Specificity Of The Chloroplastic N-Recognin Atclps1 With Fluorescent Probes Bearing Different N-Terminal Ends.

ROSANO, GERMÁN L; CECCARELLI, EDUARDO A; CANTOIA, ALEJO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Sociedades de BioCiencias

Proteolysis is a highly regulated process as useful proteins mustnot be inadvertently eliminated. One of the mechanisms of targetselection for degradation is recognition of N-degrons by an N-recognin.An N-degron is structural feature of the N-terminal end whichis recognized by proteins (N-recognins) that deliver the target to aprotease o mark it for degradation. In bacteria, the N-recognin ClpSrecognizes destabilizing amino acids in the N-terminus, like Phe,Tyr, Trp and Leu, binds to the substrate and presents it to the ClpAPprotease. Binding of ClpS is also enhanced by positive charges insecond position; proteins starting with Phe-Arg (FR) are degradedwithin seconds. In chloroplasts of Arabidopsis thaliana, a homologof bacterial ClpS has been found (AtClpS1) so it is believed thatproteolysis of chloroplastic proteins is similar, although experimentalevidence is lacking. Moreover, previous results by our group haveshown that the specificity of AtClpS1 is different from bacterial ClpSas positive charges in second position seem to be detrimental forrecognition. We created fluorescent probes to survey the specificityof AtClpS1, as it was done for bacterial ClpS. The N-terminal endof the green fluorescent protein was mutated to FR/FE/EE/EL andSA, being the last two N-terminal ends from two proposed naturalsubstrates. The fluorescent probes were obtained in recombinantform and purified to homogeneity. By pull down assays and fluorescenceanisotropy experiments, we show that AtClpS1 can bind tosubstrates with negative charges in the first and second position.This is the first experimental evidence of an N-recognin of the ClpSfamily with predilection for negative charges at or near the N-terminalend. Our results will help elucidating the sequence determinantsof substrate recognition for degradation of chloroplastic proteins.